Shedding of aged photoreceptor outer segment tips (OS) and their phagocytosis by the retinal pigment epithelium (RPE) take place daily in the retina. Failure of RPE cells to phagocytose OS causes retinal dystrophy in rodents and humans. Furthermore, delayed OS degradation may contribute to development or progression of age-related macular degeneration. It is thus important to understand the molecular mechanisms of outer segment renewal. We have identified a signaling pathway in intact retina that is required to clear shed OS promptly. Lack of synchronized phagocytosis in mice deficient in alpha-v-beta5 integrin receptors causes age-related photoreceptor dysfunction and lipofuscin accumulation. Integrin signaling requires ligand engagement of integrin receptors. We thus hypothesize that timely appearance or recruitment of integrin ligand/s serves to stimulate rhythmic signaling in the retina. No such integrin ligand has been identified to date. Our preliminary data suggest the secreted integrin ligand protein MFG-E8 as candidate for this function. MFG-E8 mediates integrin-dependent phagocytosis of apoptotic cells, a process similar to OS phagocytosis. MFG-E8 is expressed by the RPE and participates in RPE phagocytosis in vitro. This exploratory project will achieve two specific objectives: Aim 1 will explore visual function, retinal morphology, in vivo and in vitro RPE phagocytosis and phagocytic signaling in a new mouse model that lacks MFG-E8. These experiments will determine if MFG-E8 functions as a necessary ligand for av|35 integrin in the retina. Aim 2 will investigate if MFG-E8 activity in the sub-retinal space correlates with avB5 ihtegrin's activation, daily OS shedding and phagocytosis. It will further explore if photoreceptor tips destined for shedding expose phosphatidylserine to increase MFG-E8 binding sites. We will combine molecular biology, microscopy, and biochemistry to detect MFG-E8 transcription, protein expression, and protein localization in mouse retina in relation to the time course of outer segment renewal. We will develop novel affinity assays to quantify phosphatidylserine exposure and MFG-E8 binding at the time of OS shedding in intact retina. The results of this exploratory project are an important prerequisite for comprehensive studies of changes in photoreceptor tip and secreted proteins that promote the timely outer segment renewal that is essential for long-term retinal function.